| Objective:ALD is due to long-term drinking or short-term drinking large quantity of ethanol,which induced liver injury.The etiology of ALD is distinct,but its pathogenesis is poorly understood to us.In the early stage,ALD model was created by intragastric injection with ethanol.Then it's demonstrated that sirtuin1,SFRS10 m RNA and protein expression level was decreased gradually after ethanol consumption.Lipin-1 can be formed to different isoforms through alternative splicing by SFRS10.Then the AML-12 cells were cultured with gradient ethanol and the expressions of sirtuin1 reduced through transfection techniques.It's found that lower expression of sirtuin1 could lead to decrease the expression of SFRS10,while the total Lipin-1 and Lipin-1? expression level increase,moreover the Lipin-1? were decreased significantly.Knocking down the expression of SFRS10 of the AML-12 cell with SFRS10 si RNA,then oil red O staining cells were observed to explore lipid deposition.The influence of decreasing SFRS10 to sirtuin1 and Lipin-1 will be explained in this study.Methods:The recovery AML-12 cells of mouse were plated and cultured at 5% CO2,37? in 12-well culture plates.When cultured to the logarithmic growth phase,the SFRS10 gene was knockdowned,respectively divided to blank group,SFRS10 si RNA null vector group,SFRS10 si RNA group and SFRS10 si RNA+Ethanol group.Then SFRS10 si RNA were transfected into SFRS10 si RNA group and SFRS10 si RNA+Ethanol group,and negative unrelated control sequence to SFRS10 si RNA null vector group,and equal volume lipofectime 2000 to blank group.Fresh complete medium was replaced after 4-6 hours,and 120 m M ethanol was added to SFRS10 si RNA+Ethanol group after 18-24 hours,while the others added with the same volume of DMEM.The cells were collectedafter 24 hours at last.RT-q PCR and Western blot were used to detect m RNA and protein expression level of SFRS10,sirtuin1,Lipin-1,Lipin-1? and Lipin-1?;and fixed by 4% paraformaldehyde to observe lipid deposition in cells by oil red O staining.Results:1 Knocking down the expression of SFRS10,and then sirtuin1,SFRS10,total Lipin-1,Lipin-1 ? and Lipin-1? m RNA expression level were tested by RT-q PCR,it's found that expression level of Lipin-1,SFRS10,sirtuin1 and Lipin-1 ? are higher in normal AML-12 cells,while Lipin-1 and Lipin-1?lower;No significant changes were found in the expression level of sirtuin1 and Lipin-1,while the expression of SFRS10 in the SFRS10 si RNA group decreased obviously compared with blank group(P>0.05),but Lipin-1?expression level was significantly higher,and Lipin-1? expression level decreased(P all<0.01);Adding ethanol into the SFRS10 si RNA+ethanol group,SFRS10 and Lipin-1? expression levels decreased further(P all <0.01),and sirtuin1 also decreased(P<0.01),while Lipin-1? raised further,and Lipin-1 also increased significantly(P all<0.01);No significant difference between the expression level of each index in the SFRS10 si RNA null vector group,compared with the blank group,(P all>0.05).No obvious changes were found to the ratio of Lipin-1?/? in the SFRS10 si RNA null vector group(P>0.05),while significant increase in the SFRS10 si RNA group(P<0.01),compared with blank group,and the ratio of Lipin-1?/? further increased in the SFRS10 si RNA+Alcohol group,(P<0.01).2 Knocking down the expression of SFRS10,and the protein expression level of sirtuin1,SFRS10,Lipin-1,Lipin-1? and Lipin-1? were tested through WB.It's demonstrated that protein expression were unanimous with RT-q PCR assay results: sirtuin1,SFRS10 and Lipin-1? protein expression were higher in the normal cells,while the total Lipin-1 and Lipin-1?expression lower;Compared with the blank group,in SFRS10 si RNA group the Lipin-1? was increased significantly(P<0.01),and Lipin-1? decreased obviously(P<0.01),while the sirtuin1 and Lipin-1 showed no significantchange(P all>0.05);When SFRS10 si RNA+ethanol group was added with ethanol,the expression of SFRS10 and Lipin-1? were further decreased(P all<0.01),and the sirtuin1 aslo decreased(P<0.01),while the expression level of Lipin-1? was increased further(P<0.01),and Lipin-1 also increased significantly(P<0.01);No obvious differences to the expression level of each index in the null vector group,compared with the blank group,(P all >0.05).No obvious chanages to the ratio of Lipin-1?/? in the SFRS10 si RNA null vector group(P all>0.05),while significantly increased in the SFRS10 si RNA group(P<0.01),compared with blank group,and in the SFRS10 si RNA+Alcohol group the ratio of Lipin-1?/? further increased,(P<0.01).3 Observing the changes of lipid from different groups by oil red O staining,it's demonstrated that no obvious fatty degeneration were found between blank group and SFRS10 si RNA null vector group;And there were many red lipid droplets in SFRS10 si RNA group;While large quantity of red lipid droplets could be seen in SFRS10 si RNA+ethanol group.Conclusions:The decrease of SFRS10 expression had no significant effect on sirtuin1,which might adjust the fatty degeneration by regulating the alternative splicing of Lipin-1,changing the ration of Lipin-1?/?. |
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